Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk

Aims/hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. Methods Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency

The heritability of beta cell function parameters in a mixed meal test design

Aims/hypothesis: We estimated the heritability of individual differences in beta cell function after a mixed meal test designed to assess a wide range of classical and model-derived beta cell function parameters. Methods: A total of 183 healthy participants (77 men), recruited from the Netherlands Twin Register, took part in a 4 h protocol, which included a mixed meal test. Participants were Dutch twin pairs and their siblings, aged 20 to 49 years. All members within a family were of the same sex. Insulin sensitivity, insulinogenic index, insulin response and postprandial glycaemia were assessed, as well as model-derived parameters of beta cell function, in particular beta cell glucose sensitivity and insulin secretion rates. Genetic modelling provided the heritability of all traits. Multivariate genetic analyses were performed to test for overlap in the genetic factors influencing beta cell function, waist circumference and insulin sensitivity. Results: Significant heritabilities were found for insulinogenic index (63%), beta cell glucose sensitivity (50%), insulin secretion during the first 2 h postprandial (42-47%) and postprandial glycaemia (43-52%). Genetic factors influencing beta cell glucose sensitivity and insulin secretion during the first 30 postprandial min showed only negligible overlap with the genetic factors that influence waist circumference and insulin sensitivity. Conclusions/interpretation: The highest heritability for postprandial beta cell function was found for the insulinogenic index, but the most specific indices of heritability of beta cell function appeared to be beta cell glucose sensitivity and the insulin secretion rate during the first 30 min after a mixed meal. © The Author(s) 2011

Two Isoforms of the mRNA Binding Protein IGF2BP2 Are Generated by Alternative Translational Initiation

IGF2BP2 is a member of a family of mRNA binding proteins that, collectively, have been shown to bind to several different mRNAs in mammalian cells, including one of the mRNAs encoding insulin-like growth factor-2. Polymorphisms in the Igf2bp2 gene are associated with risk of developing type 2 diabetes, but detailed functional characterisation of IGF2BP2 protein is lacking. By immunoblotting with C-terminally reactive antibodies we identified a novel IGF2BP2 isoform with a molecular weight of 58 kDa in both human and rodents, that is expressed at somewhat lower levels than the full-length 65 kDa protein. We demonstrated by mutagenesis that this isoform is generated by alternative translation initiation at the internal Met69. It lacks a conserved N-terminal RNA Recognition Motif (RRM) and would be predicted to differ functionally from the canonical full length isoform. We further investigated IGF2BP2 mRNA transcripts by amplification of cDNA using 5′-RACE. We identified multiple transcription start sites of the human, mouse and rat Igf2bp2 genes in a highly conserved region only 50–90 nts upstream of the major translation start site, ruling out the existence of N-terminally extended isoforms. We conclude that structural heterogeneity of IGF2BP2 protein should be taken into account when considering cellular function

Common polymorphisms within the NR4A3 locus, encoding the orphan nuclear receptor Nor-1, are associated with enhanced β-cell function in non-diabetic subjects

<p>Abstract</p> <p>Background</p> <p>Neuron-derived orphan receptor (Nor) 1, nuclear receptor (Nur) 77, and nuclear receptor-related protein (Nurr) 1 constitute the NR4A family of orphan nuclear receptors which were recently found to modulate hepatic glucose production, insulin signalling in adipocytes, and oxidative metabolism in skeletal muscle. In this study, we assessed whether common genetic variation within the <it>NR4A3 </it>locus, encoding Nor-1, contributes to the development of prediabetic phenotypes, such as glucose intolerance, insulin resistance, or β-cell dysfunction.</p> <p>Methods</p> <p>We genotyped 1495 non-diabetic subjects from Southern Germany for the five tagging single nucleotide polymorphisms (SNPs) rs7047636, rs1526267, rs2416879, rs12686676, and rs10819699 (minor allele frequencies ≥ 0.05) covering 100% of genetic variation within the <it>NR4A3 </it>locus (with D' = 1.0, r²≥ 0.9) and assessed their association with metabolic data derived from the fasting state, an oral glucose tolerance test (OGTT), and a hyperinsulinemic-euglycemic clamp (subgroup, N = 506). SNPs that revealed consistent associations with prediabetic phenotypes were subsequently genotyped in a second cohort (METSIM Study; Finland; N = 5265) for replication.</p> <p>Results</p> <p>All five SNPs were in Hardy-Weinberg equilibrium (p ≥ 0.7, all). The minor alleles of three SNPs, i.e., rs1526267, rs12686676, and rs10819699, consistently tended to associate with higher insulin release as derived from plasma insulin at 30 min(OGTT), AUC_C-peptide-to-AUC_Glucratio and the AUC_Ins30-to-AUC_Gluc30ratio with rs12686676 reaching the level of significance (p ≤ 0.03, all; additive model). The association of the SNP rs12686676 with insulin secretion was replicated in the METSIM cohort (p ≤ 0.03, additive model). There was no consistent association with glucose tolerance or insulin resistance in both study cohorts.</p> <p>Conclusion</p> <p>We conclude that common genetic variation within the <it>NR4A3 </it>locus determines insulin secretion. Thus, <it>NR4A3 </it>represents a novel candidate gene for β-cell function which was not covered by the SNP arrays of recent genome-wide association studies for type 2 diabetes mellitus.</p

Novel Meta-Analysis-Derived Type 2 Diabetes Risk Loci Do Not Determine Prediabetic Phenotypes

BACKGROUND: Genome-wide association (GWA) studies identified a series of novel type 2 diabetes risk loci. Most of them were subsequently demonstrated to affect insulin secretion of pancreatic beta-cells. Very recently, a meta-analysis of GWA data revealed nine additional risk loci with still undefined roles in the pathogenesis of type 2 diabetes. Using our thoroughly phenotyped cohort of subjects at an increased risk for type 2 diabetes, we assessed the association of the nine latest genetic variants with the predominant prediabetes traits, i.e., obesity, impaired insulin secretion, and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: One thousand five hundred and seventy-eight metabolically characterized non-diabetic German subjects were genotyped for the reported candidate single nucleotide polymorphisms (SNPs) JAZF1 rs864745, CDC123/CAMK1D rs12779790, TSPAN8/LGR5 rs7961581, THADA rs7578597, ADAMTS9 rs4607103, NOTCH2 rs10923931, DCD rs1153188, VEGFA rs9472138, and BCL11A rs10490072. Insulin sensitivity was derived from fasting glucose and insulin concentrations, oral glucose tolerance test (OGTT), and hyperinsulinemic-euglycemic clamp. Insulin secretion was estimated from OGTT data. After appropriate adjustment for confounding variables and Bonferroni correction for multiple comparisons (corrected alpha-level: p = 0.0014), none of the SNPs was reliably associated with adiposity, insulin sensitivity, or insulin secretion (all p > or = 0.0117, dominant inheritance model). The risk alleles of ADAMTS9 SNP rs4607103 and VEGFA SNP rs9472138 tended to associate with more than one measure of insulin sensitivity and insulin secretion, respectively, but did not reach formal statistical significance. The study was sufficiently powered (1-beta = 0.8) to detect effect sizes of 0.19 < or = d < or = 0.25 (alpha = 0.0014) and 0.13 < or = d < or = 0.16 (alpha = 0.05). CONCLUSIONS/SIGNIFICANCE: In contrast to the first series of GWA-derived type 2 diabetes candidate SNPs, we could not detect reliable associations of the novel risk loci with prediabetic phenotypes. Possible weak effects of ADAMTS9 SNP rs4607103 and VEGFA SNP rs9472138 on insulin sensitivity and insulin secretion, respectively, await further confirmation by larger studies

Association of Ketone Body Levels With Hyperglycemia and Type 2 Diabetes in 9,398 Finnish Men

We investigated the association of the levels of ketone bodies (KBs) with hyperglycemia and with 62 genetic risk variants regulating glucose levels or type 2 diabetes in the population-based Metabolic Syndrome in Men (METSIM) study, including 9,398 Finnish men without diabetes or newly diagnosed type 2 diabetes. Increasing fasting and 2-h plasma glucose levels were associated with elevated levels of acetoacetate (AcAc) and β-hydroxybutyrate (BHB). AcAc and BHB predicted an increase in the glucose area under the curve in an oral glucose tolerance test, and AcAc predicted the conversion to type 2 diabetes in a 5-year follow-up of the METSIM cohort. Impaired insulin secretion, but not insulin resistance, explained these findings. Of the 62 single nucleotide polymorphisms associated with the risk of type 2 diabetes or hyperglycemia, the glucose-increasing C allele of GCKR significantly associated with elevated levels of fasting BHB levels. Adipose tissue mRNA expression levels of genes involved in ketolysis were significantly associated with insulin sensitivity (Matsuda index). In conclusion, high levels of KBs predicted subsequent worsening of hyperglycemia, and a common variant of GCKR was significantly associated with BHB levels

Variance of the SGK1 Gene Is Associated with Insulin Secretion in Different European Populations: Results from the TUEF, EUGENE2, and METSIM Studies

HYPOTHESIS:Serum- and Glucocorticoid-inducible Kinase 1 (SGK1) is involved in the regulation of insulin secretion and may represent a candidate gene for the development of type 2 diabetes mellitus in humans. METHODS:Three independent European populations were analyzed for the association of SGK1 gene (SGK) variations and insulin secretion traits. The German TUEF project provided the screening population (N = 725), and four tagging SNPs (rs1763527, rs1743966, rs1057293, rs9402571) were investigated. EUGENE2 (N = 827) served as a replication cohort for the detected associations. Finally, the detected associations were validated in the METSIM study, providing 3798 non-diabetic and 659 diabetic (type 2) individuals. RESULTS:Carriers of the minor G allele in rs9402571 had significantly higher C-peptide levels in the 2 h OGTT (+10.8%, p = 0.04; dominant model) and higher AUC(C-Peptide)/AUC(Glc) ratios (+7.5%, p = 0.04) compared to homozygous wild type TT carriers in the screening population. As interaction analysis for BMIxrs9402571 was significant (p = 0.04) for the endpoint insulin secretion, we stratified the TUEF cohort for BMI, using a cut off point of BMI = 25. The effect on insulin secretion only remained significant in lean TUEF participants (BMI< or =25). This finding was replicated in lean EUGENE2 rs9402571 minor allele carriers, who had a significantly higher AUC(Ins)/AUC(Glc) (TT: 226+/-7, XG: 246+/-9; p = 0.019). Accordingly, the METSIM trial revealed a lower prevalence of type 2 diabetes (OR: 0.85; 95%CI: 0.71-1.01; p = 0.065, dominant model) in rs9402571 minor allele carriers. CONCLUSIONS:The rs9402571 SGK genotype associates with increased insulin secretion in lean non-diabetic TUEF/EUGENE2 participants and with lower diabetes prevalence in METSIM. Our study in three independent European populations supports the conclusion that SGK variability affects diabetes risk